Cultured Myxococcota are predominantly aerobic soil inhabitants, characterized by their highly coordinated predation and cellular differentiation capacities. Little is currently known regarding yet-uncultured Myxococcota from anaerobic, non-soil habitats. We analyzed genomes representing one novel order (o__JAFGXQ01) and one novel family (f__JAFGIB01) in the Myxococcota from an anoxic freshwater spring (Zodletone spring) in Oklahoma, USA. Compared to their soil counterparts, anaerobic Myxococcota possess smaller genomes, and a smaller number of genes encoding biosynthetic gene clusters (BGCs), peptidases, one- and two-component signal transduction systems, and transcriptional regulators. Detailed analysis of thirteen distinct pathways/processes crucial to predation and cellular differentiation revealed severely curtailed machineries, with the notable absence of homologs for key transcription factors (e.g. FruA and MrpC), outer membrane exchange receptor (TraA), and the majority of sporulation-specific and A-motility-specific genes. Further, machine-learning approaches based on a set of 634 genes informative of social lifestyle predicted a non-social behavior for Zodletone Myxococcota. Metabolically, Zodletone Myxococcota genomes lacked aerobic respiratory capacities, but encoded genes suggestive of fermentation, dissimilatory nitrite reduction, and dissimilatory sulfate-reduction (in f_JAFGIB01) for energy acquisition. We propose that predation and cellular differentiation represent a niche adaptation strategy that evolved circa 500 Mya in response to the rise of soil as a distinct habitat on earth. Importance The Myxococcota is a phylogenetically coherent bacterial lineage that exhibits unique social traits. Cultured Myxococcota are predominantly aerobic soil-dwelling microorganisms that are capable of predation and fruiting body formation. However, multiple yet-uncultured lineages within the Myxococcota have been encountered in a wide range of non-soil, predominantly anaerobic habitats; and the metabolic capabilities, physiological preferences, and capacity of social behavior of such lineages remain unclear. Here, we analyzed genomes recovered from a metagenomic analysis of an anoxic freshwater spring in Oklahoma, USA that represent novel, yet-uncultured, orders and families in the Myxococcota. The genomes appear to lack the characteristic hallmarks for social behavior encountered in Myxococcota genomes, and displayed a significantly smaller genome size and a smaller number of genes encoding biosynthetic gene clusters, peptidases, signal transduction systems, and transcriptional regulators. Such perceived lack of social capacity was confirmed through detailed comparative genomic analysis of thirteen pathways associated with Myxococcota social behavior, as well as the implementation of machine learning approaches to predict social behavior based on genome composition. Metabolically, these novel Myxococcota are predicted to be strict anaerobes, utilizing fermentation, nitrate reduction, and dissimilarity sulfate reduction for energy acquisition. Our results highlight the broad patterns of metabolic diversity within the yet-uncultured Myxococcota and suggest that the evolution of predation and fruiting body formation in the Myxococcota has occurred in response to soil formation as a distinct habitat on earth.

Calcium (Ca2+) is well known as a second messenger in eukaryotes, where Ca2+ signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca2+ signaling, little is known about the mechanisms of bacterial Ca2+ signaling. Previously, we have identified a putative Ca2+-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca2+ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca2+ with 13.7 µM affinity, and that mutations at the +X and −Z positions within each or both EF-hand motifs abolished Ca2+ binding. We also show that the hydrophobicity of EfhP increased in a Ca2+-dependent manner, however no such response was detected in the mutated proteins. 15 N-NMR showed Ca2+-dependent chemical shifts in EfhP confirming Ca2+-binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca2+ binding abolished Ca2+ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca2+, which triggers its structural changes required for the Ca2+ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca2+ sensor.

The baculovirus  Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a pathogen of lepidopteran insects, has a striking dependence on the host cell actin cytoskeleton. During the delayed-early stage of infection, AcMNPV was shown to induce the accumulation of actin at the cortex of infected cells. However, the dynamics and molecular mechanism of cortical actin assembly remained unknown. Here, we show that AcMNPV induces dynamic cortical clusters of dot-like actin structures that mediate degradation of underlying extracellular matrix and therefore function similarly to clusters of invadosomes in mammalian cells. Furthermore, we find that the AcMNPV protein actin-rearrangement-inducing factor-1 (ARIF-1), which was previously shown to be necessary and sufficient for cortical actin assembly and efficient viral infection in insect hosts, is both necessary and sufficient for invadosome formation. We mapped the sequences within the C-terminal cytoplasmic region of ARIF-1 that are required for invadosome formation and identified individual tyrosine and proline residues that are required for organizing these structures. Additionally, we found that ARIF-1 and the invadosome-associated proteins cortactin and the Arp2/3 complex localize to invadosomes, and Arp2/3 complex is required for their formation. These ARIF-1-induced invadosomes may be important for the function of ARIF-1 in systemic virus spread. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Cultured Myxococcota are predominantly aerobic soil inhabitants, characterized by their highly coordinated predation and cellular differentiation capacities. Little is currently known regarding yet-uncultured Myxococcota from anaerobic, non-soil habitats. We analyzed genomes representing one novel order (o__JAFGXQ01) and one novel family (f__JAFGIB01) in the Myxococcota from an anoxic freshwater spring in Oklahoma, USA. Compared to their soil counterparts, anaerobic Myxococcota possess smaller genomes, and a smaller number of genes encoding biosynthetic gene clusters (BGCs), peptidases, one- and two-component signal transduction systems, and transcriptional regulators. Detailed analysis of thirteen distinct pathways/processes crucial to predation and cellular differentiation revealed severely curtailed machineries, with the notable absence of homologs for key transcription factors (e.g. FruA and MrpC), outer membrane exchange receptor (TraA), and the majority of sporulation-specific and A-motility-specific genes. Further, machine-learning approaches based on a set of 634 genes informative of social lifestyle predicted a non-social behavior for Zodletone Myxococcota. Metabolically, Zodletone Myxococcota genomes lacked aerobic respiratory capacities, but encoded genes suggestive of fermentation, dissimilatory nitrite reduction, and dissimilatory sulfate-reduction (in f_JAFGIB01) for energy acquisition. We propose that predation and cellular differentiation represent a niche adaptation strategy that evolved circa 500 Mya in response to the rise of soil as a distinct habitat on earth.

Pseudomonas aeruginosa infects patients with cystic fibrosis, burns, wounds and implants. Previously, our group showed that elevated Ca2+ positively regulates the production of several virulence factors in P. aeruginosa , such as biofilm formation, production of pyocyanin and secreted proteases. We have identified a Ca2+-regulated β-propeller putative phytase, CarP, which is required for Ca2+ tolerance, regulation of the intracellular Ca2+ levels, and plays a role in Ca2+ regulation of P. aeruginosa virulence. Here, we studied the conservation of carP sequence and its occurrence in diverse phylogenetic groups of bacteria. In silico analysis revealed that carP and its two paralogues PA2017 and PA0319 are primarily present in P. aeruginosa and belong to the core genome of the species. We identified 155 single nucleotide alterations within carP, 42 of which lead to missense mutations with only three that affected the predicted 3D structure of the protein. PCR analyses with carP-specific primers detected P. aeruginosa specifically in 70 clinical and environmental samples. Sequence comparison demonstrated that carP is overall highly conserved in P. aeruginosa isolated from diverse environments. Such evolutionary preservation of carP illustrates its importance for P. aeruginosa adaptations to diverse environments and demonstrates its potential as a biomarker.